Abstract Rap1 is a central signaling node connecting agonist stimulation to talin recruitment to integrins, a final common step in integrin activation. In leukocytes, RIAM is a Rap1 effector that mediates talin-dependent activation; however, in platelets the identity of such Rap1 effectors is obscure and is a key gap in our understanding. Preliminary data suggest a new evolutionarily-conserved paradigm that talin contains two Rap1 binding sites that enable it to serve as a Rap1 effector. The applicant hypothesizes that talin itself is the principal, and perhaps only, Rap1 effector implicated in platelet integrin activation. Secondly, he suggests that that the talin-Rap1 interaction may contribute to integrin function even in leukocytes that contain an abundant Rap1 effector, RIAM and in endothelial cells that contain substantial lamellipodin, a RIAM paralogue. To examine these ideas: Specific Aim 1 will test the hypothesis that a direct Rap1-talin interaction plays a central role in platelet integrin activation. Mice in which the megakaryocytes and platelets express talin with either or both Rap1 binding sites disabled will be analyzed for platelet structure, function, and formation. Hemostasis and thrombosis will be tested in a variety of assays. A biomembrane force probe will characterize the role of the Rap1-talin interaction in individual ?IIb?3 integrin-ligand bonds. Specific Aim 2 will test the hypothesis that direct Rap1-Talin interaction is important in RIAM-replete cells. In collaboration with Project 2 Integrin activation, conformation, and topology in leukocytes will be analyzed in cells bearing each of the three talin alleles described in Aim1. The effect of each mutation in combination with RIAM deletion or with RIAM over-expression will test the relative roles of these two Rap1 effectors. Specific Aim 3 will test the hypothesis that the talin-Rap1 interaction is important in endothelial cells. Mice expressing one of the three mutant talin alleles selectively in endothelium will be studied for hemorrhage and developmental phenotypes. Effects on integrin activation, spreading, and cell-cell junctions will be assessed in vitro in primary lung and brain microvascular endothelial cells. Lamellipodin deletion and over expression will enable an analysis of the relationship of lamellipodin and Rap1 binding to talin in endothelial cell functions. Together, achievement of these aims will test the paradigm-shifting hypothesis that talin itself serves as the major Rap1 effector in platelet function in hemostasis and thrombosis and evaluate the importance of the Rap1-talin interaction in leukocytes and endothelial cells.